Transmission electron microscopes (TEMs) can be used in a wide range of applications, such as microelectronics, materials science and biology. An overview of transmission electron microscopes can be found in Chapter 1 of D. B. Williams and C. B. Carter, Transmission Electron Microscopy, (2nd Ed., Springer, 2009).
Satisfactory imaging of so-called “weak phase objects”, such as specimens which comprise biological or other organic material, can be difficult with conventional TEMs.
For weak phase objects, virtually all of the contrast in the image arises from interference between electrons scattered by the specimen and unscattered electrons. The image intensity thus depends on the phase shift of the scattered electrons relative to the unscattered electrons and on the spatial frequency composition of the observed region of the specimen.
Within the widely used weak phase object approximation, see for example Chapter 6.2 of L. Riemer and H. Kohl, Transmission Electron Microscopy: Physics of Image Formation, (5th Ed, Springer, 2008), the contrast can be described by a phase contrast transfer function (PCTF) which depends on the imaging electron wavelength, lens aberrations, lens defocus and the spatial frequency composition of the observed sample region of the (weak phase object) sample. For a weak phase object and a focussed TEM image, the phase contrast at low spatial frequency (typically corresponding to features having length scales greater than about 3 nm) tends towards zero and is less than half of the contrast available to higher spatial frequency components. Thus, it can be difficult to distinguish phase variations which occur at low spatial frequency. However, for biological samples in particular, there is significant information of interest at these length scales.
While the contrast improves at higher spatial frequencies, for certain lens arrangements, the contrast can become inverted at higher spatial frequencies if these are allowed to pass through the imaging apertures in the system. For atomic-resolution images, this can result in atoms either appearing as black or white dots depending on their spatial separation, thereby making interpretation of the images more challenging.
To try and overcome these problems, TEM operators collect images at different defocus settings which have different phase contrast transfer functions, allowing some possible enhancement of the phase contrast at lower spatial frequencies and changing the frequency at which inversions occur at higher spatial frequencies. Using different images and image processing software, an operator provided with details of the microscope characteristics can construct a single image which allows image contrast to be interpreted more easily.
Some experienced TEM operators may even be able to interpret a series of images and gain an understanding of the phase variation within the sample without the aid of image processing software. However, the approach of collecting multiple images at different defocus settings has drawbacks. Firstly, the sample receives a higher dose of electrons. Generally, electron dose is minimized to reduce the possibility of damaging the sample. Secondly, choosing appropriate values of defocus and interpreting multiple images requires considerable skill and knowledge. Thirdly the defocused images do not permit straightforward observation of the sample at the ultimate (focused, with no contrast inversions) resolution of the microscope.
Phase plates are used as means to alleviate some of these problems by providing an enhancement of phase contrast over a range of frequencies. A review of phase plates for electron microscopes used in biological applications, where the intention is to enhance low spatial frequency phase contrast, is given by Kuniaki Nagayama: “Development of phase plates for electron microscopes and their biological application”, European Biophysical Journal, pages 345 to 358, volume 37 (2008).
One well-known phase plate is a Zernike phase plate. This advances the phase of the scattered electrons by π/2 relative to the unscattered electrons which appear near and in the central region of the back focal plane of the microscope. In its simplest form such a phase plate is formed from a continuous or perforated film placed near the back focal plane, where the central region is thinner than the surrounding region or, in the case of the perforated film, absent.
In this type of film based phase plate, some scattering inevitably occurs in the film. Thus some information from the sample object is lost when using this type of phase plate. This type of phase plate can also suffer from beam induced charge up and contamination. The phase plate also exhibits energy dependency. A further particular problem is that it can be difficult to control accurately the thickness of the film and, thus, the phase shift. Furthermore, the thickness of the film can change over time due to contamination. Further details of some of these problems are detailed in R. Danev, K. Nagayama, Transmission Electron Microscopy with Zernike Phase Plate, Ultramicroscopy 88, 243 (2001).
A Zernike style phase plate using an electrostatic Einzel lens arrangement in the central region to achieve the π/2 phase shift (as first proposed by Boersch in 1947 and also known as a “Boersch phase plate”) overcomes some of these problems. However, this phase plate still suffers some disadvantages. In particular, the support structure for the central electrostatic lens tends to block certain spatial frequency components. Furthermore, the phase contrast transfer function is only enhanced above a certain spatial frequency (typically about (⅙) nm−1) due to the finite size of a central lens. Moreover, the phase shift varies with electron energy. Additionally, the phase plate has a large cross-sectional area and so is prone to contamination (particularly on the rim around the unscattered beam focus) and to charging up. The phase plate is more complex to fabricate and install since it requires electrical contacts to electrodes. Also, once installed, the phase plate voltages need to be tuned for specific beam energies, as detailed by for example E. Majorovits, B. Barton, K. Schultheiss et al., Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate, Ultramicroscopy 107, 213 (2007).
Another known type of phase plate is a Zernike-style phase plate with a magnetic central ring which provides a π/2 phase shift at the centre of the beam near the back focal plane, such as in Japanese patent application 58-112718 (1985). This structure too suffers several disadvantages. Similar to its electrostatic counterpart, the phase contrast transfer function is only enhanced above certain spatial frequency due to the finite size of central aperture. This phase plate too is prone to contamination, particularly around the rim surrounding the central unscattered beam. In addition, similar to its electrostatic counterpart, the support structure blocks certain spatial frequency components.
Another form of phase plate is the Hilbert-type phase plate. This type of phase plate creates a π phase shift over a half plane at or near the back focal plane (BFP) or a plane conjugate to the BFP. As with the Zernike phase plate, this can be realized with a thin film to create the π phase shift over the half plane, but this has similar disadvantages of charge-up, contamination, loss of information, energy dependency, and difficulty to control accurately the thickness of the film. Further, the resulting image appears as if illuminated from a specific direction, with shadowing artefacts appearing in the image.
A Hilbert-type phase plate using magnetic bar spanning the centre of the BFP overcomes some of these problems. However, for optimum contrast to be gained from this type of phase plate, the magnetic bar must be placed very close the central unscattered beam, and may even directly intersect the unscattered beam. This can lead to contamination and charge up. As with the images produced from all Hilbert phase plates, the resulting images have a characteristic of appearing illuminated from one side, with shadow-like artefacts appearing in the image.